Villin is a Ca2+ dependent actin bundling protein found in brush border microvilli of absorptive cells. Tight tissue-specific control of villin expression is apparent very early in mouse embryogenesis. on day 6, villin can be detected in yolk sac visceral (but not parietal) endoderm. Further differentiation is accompanied by an increase in villin concentration and redistribution of the protein to the apical surface of the cell. Expression in the embryonic gut is first detected on day 8.5 and continues in the adult intestine. Again, changes in villin concentration and distribution precede the development of a fully differentiated brush border. This proposal is designed to test two hypotheses: a) that specific cis-acting sequences and their associated transacting factors mediate the tissue-specific expression of the villin gene, and b) that this strict regulation is necessary because the villin molecule is required for morphogenesis of the brush border. Initial experiments will identify the cis elements necessary for the characteristic pattern of villin expression. The chromatin surrounding the villin gene will be scanned for DNasel hypersensitive sites which correlate with villin expression. Enhancer trap assays will be combined with assays for promoter function in HT-29, CaCo-2 and mouse F9 (embryonal carcinoma) cells to determine the minimal promoter and enhancer elements necessary for villin expression. Once important regulatory elements are defined, their interactions with nuclear proteins from a variety of tissues will be tested in footprinting and band shift assays. These studies could lead to the identification of intestine- specific or visceral endoderm-specific transactivators. To study the role of villin the morphogenesis of the brush border, three model systems will be utilized. Intestinal morphogenesis will be studied in a mouse mutant, Splotch retarded, which carries a deletion on chromosome 1 that includes the villin gene. To complement and extend these studies, a specific knockout of one villin allele will be produced by homologous recombination in embryonic stem cells. The effect of the knock-out will be studied in differentiating embryoid bodies, in developing transgenic mice produced by blastocyst injection of the knockout cells and in mice which are null for villin, produced by brother-sister mating of the transgenic knockout line. Finally, antisense technologies will be used to reduce or ablate villin message in differentiating F9 and HT-29 cells, where the differentiation process is accompanied by the formation of a brush border. In all of these systems, the possibility that villin- induced changes in cell shape ,affect the proper apical distribution of the brush border hydrolases will be explored.